Although covalent damage to proteins can be caused by numerous reactions involving the side chains of amino acids and the amide linkages of the polypeptide backbone, it has recently been discovered that the deamidation of asparagine side chains at specific sites is a major source of spontaneous structural damage to peptides and proteins in the pH range to which proteins are most typically exposed [Aswad et al., Adv. Exper. Med. Biol. 231, 247-259 (1988); Johnson et al., Arch. Biochem. Biophys. 268, 276-286 (1989)]. For example, there is evidence that deamidation of asparagine is primarily responsible for the inactivation of lysozyme, triosephosphate isomerase, adrenocorticotropin, and calmodulin. In small peptides, deamidation of asparagine proceeds most rapidly when the amino acid sequence favors intramolecular catalysis, and the formation of a cyclic imide intermediate. Isoaspartyl linkages can also be generated in small peptides through isomerization of aspartate [Swallow and Abraham, Biochem J. 70, 364-373 (1958); Geiger and Clarke, J. Biol. Chem. 262, 785-794 (1987)]. The mechanisms of the formation of isoaspartyl linkages upon deamidation of asparagine or isomerization of aspartate are illustrated in FIG. 1.
Until now, there has been no direct method for quantitation of the amount of isoaspartate in proteins Essentially three types of methods have been known in the art.
One method is to sequence peptides derived from a protein and look for regions where sequencing fails at a site where aspartate or asparagine would normally be found. This method has been employed to infer the presence of isoaspartate in deaminated adrenocorticotropin [Graf, et al., Acta Biochem. Biophys. Acad. Sci. Hung. 6, 415-418 (1971)]. Sequencing a single protein typically requires months to years.
A second method is to digest the protein with a variety of proteases and look specifically for dipeptides containing an isoaspartyl linkage [Haley and Corcoran, Biochemistry 6, 2668-2672 (1967); Pisano et al., Arch. Biochem. Biophys. 117, 394-399 (1966)]. This method requires several days to analyze one sample, and involves a number of specialized reagents (isoaspartyl dipeptide standards) some of which are not commercially available. This method is also far from being quantitative.
The third method is to incubate the protein in acetic anhydride and pyridine in the presence of extremely high levels of radioactive water (.sup.3 H.sub.2 O), and then subject the protein to amino acid analysis, looking for incorporation of the tritium into aspartate [Spiess, J. in Methods of Protein Microcharacterization (Humana Press), 363-377 (1986)].
Interest in isoaspartate has increased in recent years following the discovery that it is selectively methylated by a protein carboxyl methyltransferase, which is present in a wide variety of species and cell types.
Protein L-isoaspartyl methyl transferases (PIMTs) are enzymes catalyzing methyl ester formation in a broad range of proteins They have an unusual substrate specificity whereby almost any protein, especially if denatured, serves as methyl acceptor to some degree However, the methyl group incorporation is generally substoichiometric, and it appears that only a subpopulation of the molecules in a given protein are capable of functioning as methyl acceptors.
In 1984 the laboratory of Dr. Dana W. Aswad at the University of California, Irvine, and, independently, Dr. Steven Clarke's laboratory at UCLA, discovered that an enzyme then called protein carboxyl methyltransferase (PCMT) or protein methylase II or protein carboxymethylase, exhibited a highly selective methylation of L-isoaspartyl residues in peptides More particularly, it was found that this enzyme catalyzed the methylation of isoaspartate in a damaged (deamidated) form of porcine adrenocorticotropin (ACTH) [Aswad, D.W. J. Biol. Chem. 259, 10714-10721 (1984) and Murray, E.D. Jr., and Clarke, S., J. Biol. Chem. 259, 10722-10732 (1984)]. Alkaline deamidation of asparagine 25 in ACTH was described to proceed through a cyclic imide intermediate, the hydrolysis of which yielded two products, a peptide which contained a normal .alpha.-linked L-aspartyl residue and a peptide with an atypical .beta.-linked L-aspartyl residue. The methyltransferase was found to specifically recognize and methylate the unusual aspartyl residue having a .beta.-carboxyl linkage in position 25 of ACTH.
Subsequent work in these laboratories [Aswad et al., Biochemistry 26, 675-682 (1987); Johnson et al., J. Biol. Chem. 262, 5622-5629 (1987); Aswad and Johnson, Trends Biochem. Sci. 12, 155-158 (1987); Ota, et al., J. Biol. Chem. 262, 8522-8531 (1987); Ota and Clarke, J. Biol. Chem. 264, 54-60 (1989); McFadden and Clarke, J. Biol. Chem. 261, 11503-11511 (1986), and in the laboratory of Dr. Patrizia Galletti in Naples [DiDonato et al., Biochemistry 25, 8361-8368 (1986); Galletti et al., Adv. Exper. Med. Biol. 231, 229-245 (1988)] indicated that PCMT would methylate isoaspartyl sites in a variety of peptides with little dependence on the surrounding sequence [see e.g. Aswad and Johnson, Supra].
More recently [Henzel et al , J. Biol. Chem. 264, 15905-15911 (1989)], this enzyme has been referred to as a protein L-isoaspartyl methyltransferase (PIMT), so as to distinguish it from other protein carboxyl methylating enzymes which do not methylate isoaspartate. Accordingly, hereinafter we shall use this designation.
Existing practice of using PIMT to identify isoaspartate in proteins has been limited to basic scientific applications.
Ota and Clarke, J. Biol. Chem. 264, 54-60 (1989) investigated the formation of D-aspartyl and L-isoaspartyl (.beta.-aspartyl) residues and their subsequent methylation in bovine brain calmodulin by PIMT. They subjected intact calmodulin to methylation using partially purified PIMT, and subsequently digested the methylated calmodulin by proteolysis and separated the peptides by HPLC. Because the methylation is performed on the intact-folded protein, any isoaspartates that are buried in the interior of the protein, or held in a rigid conformation, may not be accessible to methylation by PIMT. Moreover, proteolytic digestion of the pre-methylated protein may result in significant loss of the methyl esters We have experimentally found that, for these reasons, this method severely underestimates the amount of isoaspartate in the target protein.
Di Donato et al., Biochemistry 25, 8361-8368 (1986) suspected the presence of isoaspartate in a form of ribonuclease (RNAse) They digested the protein with trypsin, then separated the resulting peptides by HPLC. A peptide which was suspected of harboring the isoaspartate was isolated and tested for its ability to accept methyl groups upon incubation with PIMT and radiolabeled S-adenosyl-L-methionine (AdoMet). This approach is suitable for analysis of specific peptides derived from a protein when there is some a priori evidence for location of the isoaspartate. It is not a convenient approach for routine screening of proteins, however, because each peptide would have to be methylated separately. A given protein could result in hundreds of separate methylation reactions for a complete screening
Galletti et al., Adv. Exper. Med. Biol. 268, 229-245 (1988) have used PIMT to identify isoaspartate in mouse epidermal growth factor. The protein was treated in a manner which was expected to generate isoaspartate. They then unfolded the protein by reducing and chemically blocking the disulfide bonds which normally serve to stabilize the native structure. The unfolded form of EGF showed evidence of significant levels of isoaspartate. Control experiments indicated that the unfolding per se did not generate the isoaspartate, but did allow its detection. A possible problem with this method is that sulfhydryl modification may not always be effective at rendering all possible sites accessible to methylation. Another problem is that the conditions employed for blocking the sulfhydryl groups may inadvertently introduce isoaspartate into the protein leading to artifacts.
In proteins lacking disulfide bonds, this approach is of no use.
Aswad et al., J. Cell. Biochem. Supplement 13A, UCLA Symposia on Molecular & Cellular Biology, 18th Annual Meetings, Abstracts, A 202, p. 65 (1989) report the selective methylation of calmodulin and human growth hormone after digestion with trypsin. The peptide fragments were separated by HPLC, and assayed for methyl incorporation.
There is no commercially applicable method known in the art utilizing PIMT for analysis of isoaspartate in proteins.